Fanconi Anemia (FA) is an autosomal recessive disease characterized by progressive pancytopenia, cancer susceptibility, and multiple congenital abnormalities. Cells from patients with FA are hypersensitive to the DNA- damaging agents. mitomycin C (MMC) and diepoxybutane (DEB), and the molecular defect is presumably one of DNA repair. A cDNA clone (Strathdee et al. Nature 356: 763-767, 1992). which corrects one of the four known Fanconi Anemia complementation groups in vitro has recently been isolated. The function of the 60 kD FACC polypeptide remains unknown. Using the recently cloned human Fanconi Anemia Complementation Group C (FACC) cDNA and a newly developed antiserum against the FACC polypeptide, we propose to study the molecular pathogenesis of FA. Project I, during the proposed five year study period, will be the delineation of functional domains of the FACC polypeptide by in vitro mutagenesis. Using a functional assay, the ability of the wild-type FACC cDNA to correct cellular sensitivity of HSC536N cells to MMC, we will delineate domains of the FACC important for this cellular function. In vitro synthesized and naturally occurring FACC mutants will be assayed for (a) correction of MMC sensitivity and (b) cellular localization (by immunofluorescence). Project II will focus on the identification and cloning of FAP-50, an FACC-associated protein of 50 kD which forms a stable protein complex with FACC. The identification of this protein should help elucidate the cellular role of the FACC polypeptide. Project III will investigate the role of the FACC polypeptide as a cell cycle regulator and as a tumor suppressor. The expression of FACC mRNA and polypeptide will be assayed in multiple cell lines, at different times during the cell cycle, and in various leukemias and cancer cells.